A majority of the over 300 tested N-nitroso compounds have been positive for carcinogenicity in laboratory animals (Preussmann, R. et al, "Chemical Carcinogens, Second Edition"; Searle, C. W., Ed.; American Chemical Society: Washington. D.C. 1984; American Chemical Society Monograph No. 182; Chapter 12). Humans are exposed to N-nitroso compounds from a variety of sources including foods, occupational exposures, cosmetics, and formation within the body. Certain N-nitrosamides are also widely used as therapeutic anticancer agents (Preussmann, R. et al, "Chemical Carcinogens, Second Edition"; Searle, C. W., Ed.; American Chemical Society: Washington, D.C., 1984; American Chemical Society Monograph No. 182; Chapter 13; Hotchkiss, J. H. Advances in Food Research 31,54 (1987)). For these reasons, there is considerable interest in the analysis of trace levels of these compounds in biological and environmental media.
Many N-nitrosamines can be analyzed, either directly or after derivatization, by gas chromatography coupled to a Thermal Energy Analyzer Detector (TEA; Hotchkiss, J. H. J. Assoc. Offic. Anal Chem., 64:1037 (1981)). The TEA is a modified chemiluminescence detector which relies on the thermal cleavage of the N-N bond producing a nitric oxide (NO) radical. The nitric oxide is reacted with ozone to produce an excited nitrogen dioxide which emits a photon upon decay (Fine, D. H. et al, J. Chromatog., 107:351 (1975)). The photons are detected and amplified by a photomultiplier tube. There are two limitations to this system; first is that the N-nitroso compounds must be volatile enough, or made volatile enough for gas chromatography, and secondly, they must yield nitric oxide upon thermolysis. N-Nitrosamides and related compounds, unlike N-nitrosamines, typically rearrange upon thermolysis to yield molecular nitrogen instead of nitric oxide and are only weakly detected by the TEA. In addition, several N-nitrosamines that are of interest are not suitable for gas chromatography. HPLC-TEA methods have been reported (Sen. N.P. et al, Food Additives Contamin., 4:357 (1987)) but mobile phases containing water give inconsistent results.
Shuker and Tannenbaum, Anal. Chem. 55:2152 (1983) have described a method in which N-nitrosamides are photolytically cleaved by uv irradiation. The resulting nitric oxide was oxidized to nitrite, and reacted post-column with Griess reagent to form a chromophore which was detected spectrophotometrically at 541 nm. Sensitivity was 6 to 100 ng as injected depending on the specific N-nitroso compound.
Fine et al, In "The Relevance of N-nitro Compounds to Human Cancer"; Bartsch, H.; O'Neill, I.; Schulte-Hermann, R., Ed.; International Agency For Research on Cancer: Lyon, 1987; IARC Scientific Publication No. 84; p. 216 have reportedly modified the pyrolysis chamber in a standard TEA in order that N-nitrosamides release nitric oxide during pyrolysis. Sensitivities (3:1; S:N) of less than 1 ng injected were reported for standards. Complete details of the instrument were not provided and the need for further development was noted. Singer et al, J. Chromatography, 133:59 (1977) used dilute acid to cleave N-nitrosamides and coupled the resulting nitrite to Griess reagent. A sensitivity of 50 to 100 ng was reported. Sen and Seaman. In "N-nitro Compounds: Occurrence, Biological Effects and Relevance to Human Cancer"; O'Neill, I. K.; Von Borstel, R. C.; Miller, C. T.; Long, J.; Bartsch, H., Ed.; International Agency for Research on Cancer: Lyon, 1984; IARC Scientific Publication No. 57; p. 137, have also chemically cleaved the nitroso group from N-nitroso compounds and have detected the nitric oxide by chemiluminescence using a modified TEA. The slow response time of the system caused considerable peak broadening. Detection of N-nitrosamides by uv (Krull, I. S., et al, J. Anal. Toxicol., 5:42 (1981)), MS after derivatization (Weinkam, R. J. et al, Clin. Chem., 24:45 (1978)), and denitrosation with subsequent detection of the amide (Mirvish, S. S. et al, J. Agric. Food Chem., 28:1175 (1980)) have also been proposed.
U.S. Pat. No. 4,233,030 to Twitchett et al teaches a photochemical reaction interposed between a high pressure liquid chromatograph column and a detector where the analysis is based on a constituent or a species resulting from irradiation. The property detected is either an increase or a decrease in fluorescence in compounds such as lysergic acid diethylamide (LSD) and cannabinol when subjected to visible or uv light absorbance. The property of the converted product that is analyzed is either enhanced light absorbance or reduced light absorbance.
The difficulty of analyzing N-nitroso compounds is noted by Fine et al in U.S. Pat Nos. 3,996,002; 3,996,003; 3,996,004; and 3,973,910, all of which are incorporated herein by reference to show both the problem faced and earlier attempts to solve it by interposing a "non catalytic pyrolysis" step between the liquid chromatograph and the detector means. U.S. Pat. No. 3,996,003 and 3,996.004 relate to the detection of NO and nitrate and nitrite respectively. U.S. Pat. No. 3,996,008 and 3,996,009 related to similar processes using a gas chromatograph. In U.S. Pat. No. 3,973,910 Fine converts the nitroso compounds to NO gas by the sole energizing step of heating at 200.degree. C. to 300.degree. C.
All of the above methods fail to satisfy the need for a highly selective, sensitive and simple process for the resolution, dection and analysis of N-nitroso compounds including N-nitrosamides and non-volatile N-nitrosamines in nanogram quantities. The present apparatus and methods fills such a need.